Insect Science (2019) 26, 466–478, DOI 10.1111/1744-7917.12551 ORIGINAL ARTICLE Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella Valeria Mereghetti1, Bessem Chouaia1, Lidia Limonta2, Daria Patrizia Locatelli2 and Matteo Montagna1 1Dipartimento di Scienze Agrarie e Ambientali, Universita` degli Studi di Milano, Milan, Italy and 2Dipartimento di Scienze per gli Alimenti la Nutrizione, l’Ambiente, Universita` degli Studi di Milano, Milan, Italy Abstract Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we char- acterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities world- wide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversi- fied microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approx- imately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (64%), followed by Propionibacterium, Delftia, Pseudomonas,andStenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were as- signed to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indi- cates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages. Key words Burkholderia; Illumina sequencing; Indian meal moth; insect symbiosis; Proteobacteria Introduction culture-independent approaches based on high through- put sequencing became a powerful method to investigate All animals, including insects, harbor a multitude of the microbial community associated with insects in a microorganisms (bacteria, protozoa and fungi) (McFall- wide taxonomic range of hosts (Chakravorty et al., 2007; Ngai et al., 2013). In particular the successful adaptation Huse et al., 2008; Wang & Qian, 2009). In most of the of insects reflects the very close and ancient mutualistic cases, microbiota investigation highlighted interesting association that these groups of animals shared with host–symbiont interactions, representing key aspects in the microorganisms (Douglas, 2014). In recent years, insect’s survival and adaptation, such as development, nutrition, detoxification, and maintaining of the immune Correspondence: Matteo Montagna, Dipartimento di system (Dillon & Dillon, 2004; Engel & Moran, 2013). Scienze Agrarie e Ambientali, Universita` degli Studi di Mi- Regarding lepidopterans, the studies on their bacterial lano, via celoria 2, Milan, 20133, Italy. Tel: +39 02 503 16782; communities have increased in the recent years, espe- fax: +39 02 503 16781; email: [email protected] cially for insect pest moths such as Ostrinia nubilalis 466 C 2017 Institute of Zoology, Chinese Academy of Sciences P.interpunctella microbiota: from eggs to adults 467 (Belda et al., 2011), Spodoptera littoralis (Tang et al., Materials and methods 2012), Lymantria dispar (Broderick et al., 2004; Lin et al., 2015), Helicoverpa armigera (Xiang et al., 2006), Ethics statement Plutella xylostella (Xia et al., 2013), and Bombyx mori (Liang et al., 2014). Furthermore, some of these studies P.interpunctella are not listed in any national or regional revealed the role played by the bacteria in helping the host law as protected or endangered species. in a broad range of activities. As a matter of fact, Ente- rococcus sp. and Staphylococcus sp. provide their hosts, Insect rearing and sampling Hyles euphorbiae and Brithys crini (two lepidopterans feeding on plants producing alkaloids and latex), resis- The specimens used in this study were obtained from a tance to these toxic compounds (Vilanova et al., 2016). A P.interpunctella population maintained in the insectary at further symbiont-derived benefit regards the production DEFENS (Department of Food, Environmental and Nu- of antimicrobial peptides, which protect the insect host tritional Sciences—Universita` degli Studi di Milano) and against the invasion of foreign bacteria (e.g., Shao et al., reared under controlled conditions of temperature (26 ± 2017). Almost all the previously reported studies focused 1 °C), humidity (RH = 65% ± 5%) and a 16 h : 8 h on a single developmental stage (e.g., third instar larvae, light : dark photoperiods. Insects were routinely fed on Hernandez-Flores` et al., 2015; Snyman et al., 2016; the an artificial diet following Stampini and Locatelli (2007), adult stage, Montagna et al., 2016), ignoring the changes with some modifications (20% increase of honey content that may occur of the microbiota structure along the life and without beer yeast). In order to obtain eggs, adult fe- cycle of these holometabolous insects. To our knowledge, male were separated from the main laboratory population only two studies have investigated the microbiota’s and allowed to oviposit for 3 h. Ninety eggs were directly structure across the life cycle in lepidopterans. The first stored at –80 °C without sterilization, while the remaining investigation was reported by Hammer et al. (2014) eggs were allowed to hatch. The first-instar larvae, 3 h af- and revealed that larvae and adults of the neotropical ter the emergence from the egg, and the last-instar larvae butterflies Heliconius erato harbored a taxonomically (before pupation) were collected and stored in absolute different microbiota. A similar result was observed in the ethanol at –80 °C. Last-instar larvae were sexed (Hamlin cotton leafworm Spodoptera littoralis (Chen et al., 2016). et al., 1931) and then let to molt separating males from The Indian meal moth (IMM) Plodia interpunctella females. Once emerged, adults were stored in absolute (Hubner)¨ represents one of the major pests of food com- ethanol at –80 °C. modities worldwide (Vick et al., 1986; Cuperus et al., 1990; Rees, 2004). The economic impact of this moth is very high, because it feeds on a wide range of stored Sample processing and DNA extraction products, such as grains, chocolate, nuts, powdered milk, and dry meats (Perez-Mendoza & Aguilera-Pena, 2004); Three replicates, for each developmental stage, were damages are represented by larval silk webs and frass (Fa- processed as described below. Each replicate was com- sulo & Knox, 2009). In a previous study the microbiota posed of five specimens from the same developmen- associated with IMMs reared under different diets was tal stage. The collected specimens (except the eggs) investigated (Montagna et al., 2016). Two distinct ento- were surface-sterilized using previously published pro- motypes were discovered associated with IMMs based on tocol (Montagna et al., 2015a). Briefly, the sterilization their diet, namely protein-rich versus carbohydrate-rich, protocol consists of the following steps: (i) washing the suggesting the diet as a major driver in shaping the bac- samples with distilled water; (ii) washing with a sodium terial community (Montagna et al., 2016). hypochlorite solution at 4% for 3 min; (iii) washing with In this study, we used high-throughput sequencing a solution of PBS and 0.1% Triton-X-100 for 3 min; and technology to investigate the microbiota of IMMs in (iv) a final washing with sterile distilled water. These steps different developmental stages, namely eggs, the first- were repeated three times. and last-instar larvae, male and female adults. The The wings were removed from adults prior to sterilizing aims of this study are: (i) estimate the shift in the the body. Total bacterial DNA was extracted, under sterile IMM-associated microbiota during selected stages of its conditions, using the classical phenol–chloroform meth- life cycle and in the case of bacterial community changes; ods (Doyle & Doyle, 1990) with the following modifica- (ii) evaluate the presence of a bacterial core throughout tions: First, 500 μL of 2% CTAB (2% CTAB, 0.2% ascor- the developmental stages, and (iii) predict the functional bic acid, 1.5% PVP, 1.4 mmol/L NaCl, 20 mmol/L EDTA profiles of the bacterial communities. and 100 mmol/L Tris-HCl, pH 8.0) was added to each C 2017 Institute of Zoology, Chinese Academy of Sciences, 26, 466–478 468 V.Mereghetti et al. sample. Tissues were then disrupted using glass beads Bacterial community analysis (ø 0.1 mm) with the PrecellysR 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and in- The OTUs table obtained by QIIME, representing the cubated at 65 °C for 15 min to inactivate nucleases. After bacterial communities associated with each of the ana- centrifugation, the supernatant was incubated overnight lyzed samples, was used as an input for of the diversity with 20 μL of proteinase K (20 mg/mL) at 56 °C. In order indices estimation and following analyses. The package to purify